The technology of optical microscopes dates back centuries, and there have been developed various kinds of microscopes. In recent years, with advances in peripheral technologies including laser technology and electronic image processing technology, microscope systems of even higher functional capability have been developed.
Under such context, there has been proposed a microscope of high functional capability that can even perform a chemical analysis, as well as contrast control of an obtained image, using a double resonance absorption process caused by irradiating a sample with lights of different wavelengths (see, for example, Patent Document 1 listed below).
With this microscope, a specific molecule is selected using a double resonance absorption process, and an absorption and a fluorescence caused by a specific optical transition are observed. The principle used will be explained below with reference to FIGS. 12 to 15. FIG. 12 is a diagram of an electronic structure of valence orbits of a molecule contained in the sample. First, an electron in a valence orbit of a molecule in a ground state (S0 state) shown in FIG. 12 is excited by a light having a wavelength λ1 and thereby brought to a first electron-excited state (S1 state) shown in FIG. 13. Then, the electron is excited by a light having another wavelength λ2 in the same way and thereby brought to a second electron-excited state (S2 state) shown in 14. In this excited state, the molecule emits fluorescence or phosphorescence and thereby returns to the ground state, as shown in FIG. 15.
In the microscopy using a double resonance absorption process, an image of absorption or luminescence is observed using the absorption process as shown in FIG. 13 or the luminescence of fluorescence or phosphorescence as shown in FIG. 15. In this microscopy method, first of all, a molecule contained in the sample is excited to the S1 state as shown in FIG. 13 with a light, such as a laser beam, having the resonance wavelength λ1. On this occasion, the number of molecules in the S1 state per unit volume increases proportionally to the intensity of the irradiated light.
Here, the linear absorption coefficient is defined as a product of the absorption cross-sectional area per molecule and the number of molecules per unit volume. Thus, in a process of excitation as shown in FIG. 14, the linear absorption coefficient with respect to the resonance wavelength λ2 of the second light depends on the intensity of the first light having the wavelength λ1. In other words, the linear absorption coefficient with respect to the wavelength λ2 can be controlled with the intensity of the light having the wavelength λ1. This means that, by irradiating lights having the wavelength λ1 and the wavelength λ2, respectively, to the sample and capturing the transmitted image with respect to the wavelength λ2, it is possible completely to control the contrast of the transmitted image with the light having the wavelength λ1.
Further, if the returning process from the excited state with fluorescence or phosphorescence is possible in the excited state of FIG. 14, the intensity of luminescence is proportional to the number of molecules in the S1 state. Consequently, it is possible to control the contrast of an image also in the case of application as a fluorescence microscope.
Further, in the microscopy using a double resonance absorption process, it is also possible to perform a chemical analysis, in addition to the above-mentioned control of the image contrast. More specifically, since the outermost valence orbit shown in FIG. 12 has an energy level inherent to each molecule, the wavelength λ1 varies according to the kind of molecule and the wavelength λ2 also becomes inherent to the molecule.
By using only a single wavelength in a conventional manner, it is also possible to observe an absorption image or a fluorescence image of a specific molecule to a certain degree. In this instance, however, an accurate identification of the chemical composition of the sample is not possible because some molecules generally have an overlapped range of the absorption wavelength.
On the contrary, in the microscopy using a double resonance absorption process, molecules are limited to those that absorb a light or produce a luminescence with two wavelengths λ1 and λ2, and it is thus possible to achieve even more accurate identification of chemical composition of the sample, as compared to the prior art. Further, when exciting a valence electron, since only a light having a certain electric field vector relative to the axis of the molecule is strongly absorbed, it is possible to identify even the orientation of the same molecule by suitably determining the polarizing directions of the lights having the wavelengths λ1 and λ2 and capturing the absorption or fluorescence image.
Furthermore, a fluorescence microscope using a double resonance absorption process and having a spatial resolution higher than the diffraction limit has recently been proposed (see, for example, Patent Document 2 listed below).
FIG. 16 is a schematic diagram of a double resonance absorption process in a molecule, in which the molecule in the ground state S0 is excited to the first electron-excited state S1 with a light having the wavelength λ1 and further to the second electron-excited state S2 with a light having the wavelength λ2. In addition, FIG. 16 illustrates that fluorescence from S2 state is extremely weak for certain type of molecule.
In the case of a molecule having an optical property as shown in FIG. 16, a phenomenon of significant interest can be observed. FIG. 17 is a schematic diagram of a double resonance absorption process similar to FIG. 16, in which the horizontal axis (X axis) represents a stretch of spatial distance, which is divided into spatial regions A1 exposed to the light of the wavelength λ2 and a spatial region A0 not exposed to the light of the wavelength λ2.
In FIG. 17, a great number of molecules are excited to S1 state by the light of the wavelength λ1 in the spatial region A0, so that a fluorescence of a wavelength λ3 from the spatial region A0 is observed. In the spatial regions A1, on the other hand, due to the light of the wavelength λ2 being irradiated, most of the molecules in S1 state are instantaneously excited to the higher energy level S2 state, so that there exist no molecules in S1 state. Such a phenomenon has been confirmed as to some molecules. Since fluorescence of the wavelength λ3 is completely eliminated in the spatial region A1, and as there is no fluorescence from S2 state, fluorescence is entirely inhibited in the spatial regions A1 (fluorescence inhibiting effect). Thus, there is produced fluorescence only from the spatial region A0.
Considering the present application field of a microscope, those phenomena have an extremely important meaning. In other words, with the conventional scanning laser microscope or the like, wherein laser beam is focused into a micro beam by a condenser lens to scan over a sample to be observed, the size of the micro beam is limited within a diffraction limit determined by the numerical aperture of the condenser lens and the wavelength of the laser beam, so that a spatial resolution higher than the diffraction limit cannot theoretically be expected.
In the case of FIG. 17, however, a spatial resolution can substantially be improved because, as to the irradiated region of a light of the wavelength λ1, for example, a fluorescence region can be made smaller than the diffraction limit determined by the numerical aperture of condenser lens and the wavelength of the light by suitably and spatially combining two kinds of lights, i.e., a light of the wavelength λ1 and a light of the wavelength λ2. In the following description, the light of the wavelength λ1 is referred to as a “pump light”, and the light of the wavelength λ2 is referred to as an “erase light”. Therefore, by applying this theory, it is possible to realize a super-resolution microscope, for example a super-resolution fluorescence microscope, having a higher resolution than a diffraction limit, and using the double resonance absorption process.                Patent Document 1: JP 08-184552 A        Patent Document 2: JP 2001-100102 A        